In bacteria, DNA methylation has been thought to function primarily in restriction-modification systems. However, characterization of the Dam MTase in E. coli
showed that methylation also plays a regulatory role in the cell, including control of DNA replication and transcription (17
). Studies of the cell cycle-regulated CcrM MTases of Caulobacter
, and Brucella
suggest that bacteria belonging to the alpha subdivision of the Proteobacteria
also use adenine methylation as a regulatory mechanism (23
Here, we studied an E. coli
DNA adenine MTase from a member of the gamma subdivision of the Proteobacteria.
This enzyme is an essential, cell cycle-regulated orphan MTase that plays a similar role in regulating cell cycle events and exhibits 50% homology with a group of ortholog MTases from members of the alpha subdivision of the Proteobacteria.
These MTases methylate the recognition sequence GANTC, as shown by the resistance of chromosomal DNA to HinfI digestion (25
). An E. coli
CcrM DNA MTase that methylates the first adenine moiety of the DNA sequence ATGCAT has now been identified. This conclusion is based on the observation that phage lambda DNA, after methylation by CcrM, is resistant to cleavage by NsiI and BfrBI, which recognize the ATGCAT sequence but have different digestion sensitivities, depending on methylation at the first or second adenine. A cognate REase is not encoded by the gene adjacent to the ccM
gene. Furthermore, we showed that the activity of a cognate REase is not present in E. coli
, because it was possible to detect unmethylated DNA during the cell cycle (Fig. ).
The methylation status of ATGCAT sites in the E. coli DNA was found to change cyclically during the cell cycle. The observed cell cycle changes in the methylation state can be attributed to the temporal transcriptional regulation of ccrM in E. coli cells. Our data show that the cellular level of ccrM mRNA also changed cyclically during the cell cycle. This suggests that the CcrM protein level changes during the cell cycle.
Evidence that expression of CcrM may affect DNA replication and cell division was obtained by analysis of E. coli
cultures in which the ccrM
gene was represented by many copies or overexpressed. The numbers of genome equivalents in these cultures were more than twice the numbers of genome equivalents when the expression of ccrM
is normal. Overexpression of ccrM
had an unexpected effect on cell morphology; many of the cells were elongated, and the cell size was irregular. A similar phenotype was observed with bacteria belonging to the alpha subdivision of the Proteobacteria
orthologs when excess amounts of the CcrM proteins were present in the cells (25
). In these bacteria, the phenotype correlated with disrupted cell division. Methylation of the promoter region in bacteria could alter the expression of genes that affect cell division. Overexpression of CcM can repress the expression of genes involved in the cell cycle and cause a delay in cell division. Such a delay may lead to the elongated appearance of the cells. The disturbing effects caused by aberrant temporal expression of the ccrM
gene suggest that CcrM has an important role in regulating the cell cycle.
It has already been established that cell cycle-regulated DNA MTase is widespread in members of the alpha subdivision of the Proteobacteria
, including Caulobacter
, and Brucella
). Here we present evidence that a temporally constrained DNA methylation activity may be a conserved means of regulating fundamental aspects of the cell cycle and that this mechanism is found in another group of bacteria, the gamma subdivision of the Proteobacteria
. It is likely that undiscovered DNA MTases in other groups of bacteria play comparable roles in regulating the cell cycle. Thus, CcrM appears to be an orphan MTase which, like Dam, provides important regulatory information in members of the gamma subdivision of the Proteobacteria
. We have found that the M.EcoKCcrM MTase has a striking degree of similarity with putative DNA MTases of pathogenic members of the gamma subdivision of the Proteobacteria
, such as E. coli
O157:H7, S. enterica
serovar Typhimurium LT2, and S. flexneri
(Table ). Thus, inhibition of the ccrM
function may prove to be lethal to such bacteria, through disruption of an essential regulatory function. CcrM may be an attractive candidate target for the development of MTase inhibitors as novel antibacterial agents for use against pathogenic bacteria. To further determine the functions of the CcrM adenine MTase in E. coli
, we are currently investigating its role in the regulation of gene expression.